Volatilizable solid phase supports for compound synthesis

ABSTRACT

A solid phase synthetic method is disclosed in which the usual solid phase synthetic steps are carried out and the spent solid phase support is reacted to form a volatilizable compound upon cleavage of the reaction product from the solid phase support. The cleaved product is then separated from the volatile compound by volatilization of that compound. Exemplary solid supports that form a volatilizable compound are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 10/286,670, filed Nov. 1,2002, now U.S. Pat. No. 7,067,623, that was a division of applicationSer. No. 09/493,902, filed Jan. 28, 2000, now U.S. Pat. No. 6,476,191,which claimed priority from application Ser. No. 60/119,204 filed Feb.5, 1999, whose disclosures are incorporated herein by reference.

TECHNICAL FIELD

The invention relates to solid phase syntheses, and more particularly tosolid phase synthesis on a synthetic support that is volatilized uponthe cleavage of the synthesized material from the support.

BACKGROUND OF THE INVENTION

The preparation of compounds using a solid phase approach was firstdescribed by Merrifield in 1963 [Merrifield, 1963, J. Am. Chem. Soc.,85:2149-2154. Since this initial seminal concept, in which a polystyrenesolid phase was used to prepare peptides, a wide range of differentsolid supports have been used (i.e., polyamides [Atherton et al., 1975,J. Am. Chem. Soc., 97:6584-6585], porous glass [Parr et al., 1974.Justus Liebigs Ann. Chem., pp. 655-666] and microchip quartz [Fodor etal., 1991, Science, 251:767-773]). While useful, these solid phasesupports all require a final cleavage step, in which the compounds(peptides, peptidomimetics, oligonucleotides, small organic molecules,various heterocycles, and the like) are cleaved from the solid phase,then separated from the spent solid support.

Where the compound of interest can be used in an immobilized manner(i.e., it remains on the solid support in its final use and/ormanifestation), then the remaining solid support may not be problematic,and in fact may be useful for certain assays. However, in the majorityof cases, the compound of interest has to be used in solution andtherefore has to be separated from its solid support. Significant timeand/or cost savings would be realized if the removal of the solid phasematerial did not have to be accomplished in a separate step followingcleavage of the desired compound from the solid support (typically byfiltration or centrifugation). The invention disclosed hereinafterprovides one solution to the problem of separating the spent solidsupport from the desired synthesized material.

BRIEF SUMMARY OF THE INVENTION

The present invention contemplates solid phase synthesis on a solidsupport in which the desired product is left behind following cleavagefrom and vaporization of the solid. Thus, a solid phase synthesis methodis contemplated in which at least one reagent is coupled to a solidphase support. A plurality of reactions is carried out upon the solidphase-coupled reagent to form a solid phase-coupled reaction product,and the reaction product is cleaved from the solid phase support to forma cleaved product. The improvement in this otherwise standard synthesisis that the solid phase support is reacted to form a volatilecompound(s) that is separated from the cleaved product by vaporizationas by distillation. The desired cleaved product is preferably recoveredand the solid support is absent due to its volatilization.

A particularly preferred solid support is silica. Cleavage of theproduct from the solid support and formation of the volatile compound istypically carried out in a single step, although separate steps can beused.

The present invention has several benefits and advantages.

One benefit is the simplicity in reaction steps that are carried out inthat the usual filtering step required in prior solid phase syntheses isnot required.

An advantage of a contemplated method is that losses of desired productthat can occur because of entrapment of the desired product within theusual spent solid support or filter do not occur.

Another benefit is that the usual final extraction step(s) to remove theproduct from the solid support required in prior solid phase synthesesafter cleavage from the solid support is not required here.

Still further benefits and advantages of the contemplated invention willbe apparent to the skilled worker from the disclosure that follows.

DETAILED DESCRIPTION OF THE INVENTION

A solid phase synthetic method is contemplated in which the usual solidphase synthetic steps are carried out in the synthesis of a peptide,peptidomimetic, glycopeptide, oligonucleotide, small organic molecules,or heterocyclic product as noted hereinafter. The improvement here liesin the separation of the cleaved product from the solid support byconversion of the solid phase support into a volatile material that isseparated from the desired reaction product by vaporization so that theusually used filtration or extraction separation of the desired productfrom the spent solid phase support is unnecessary.

Thus, taking a solid phase peptide synthesis as exemplary, at least onereagent such as a side chain- and N-protected amino acid is coupled tothe solid support. A plurality of reactions is carried out on that solidphase-coupled reagent such as N-de-protection, coupling of another sidechain- and N-protected amino acid, and N-de-protecting the resultingproduct to form a solid phase-coupled reaction product. Any side chainprotecting groups present are removed, and the link between solidsupport and desired product is broken to form a cleaved product. Avolatile compound is formed from the spent solid support. In preferredpractice for peptide synthesis, HF is used to remove any side chainprotecting groups present, cleave the product from the solid support andform the volatile compound(s) from the spent solid support.

As used herein, the material formed on the solid phase support andbonded thereto is referred to as a “reaction product”. The reactionproduct can have protecting groups bonded to it or those protectinggroups can be removed.

The “cleaved product” is that material obtained upon breaking of thebond between the solid phase support and the reaction product. Thecleaved product is typically free of protecting groups but need not beso. In addition, the cleaved product is typically protonated, althoughprotonation is not a defining feature of a cleaved product.

A “spent solid support” is the material remaining after cleavage of thedesired reaction product from the support. As discussed below, the solidsupport is preferably converted into a volatile compound concomitantlywith formation of the cleaved product. In that preferred case, there isusually no spent solid support.

For example, porous glass has been used as a solid support to prepare apeptide with cleavage of the desired product effected by reaction of thesolid phase-bound peptide with methanol and triethylamine. [Parr et al.,1974, Justus Liebigs Ann. Chem., pp. 655-666.] Contrarily, using acontemplated method, the porous glass can be completely transformed byliquid or gaseous hydrogen fluoride into volatile silicon tetrafluoride(SiF₄, bp: −86° C.) that can be warmed or a vacuum applied to effectseparation, as compared to use of a reagent that cleaves the compoundfrom the support followed by filtration of the spent solid support fromthe desired compound as was carried out by Parr et al. Use of acontemplated method leaves the desired compound in the reactioncontainer, with the porous glass solid support volatilized away as SiF₄.

This concept greatly facilitates the production of individual compoundsor mixtures of compounds, or the large scale production of individualcompounds, arrays of compounds, or combinatorial libraries of mixtures[Plunkett et al., 1995, J. Org. Chem., 60:6006-6007; Houghten, 1985,Proc. Natl. Acad. Sci. USA, 82:5131-5135; Houghten et al., 1991, Nature,354:84-86; Pinilla et al., 1992, BioTechniques 13: 901-905; Ostresh etal., 1994, Proc. Natl. Acad. Sci. USA, 91: 11138-11142; Dooley et al.,1994, Science, 266:2019-2022; and Eichler et al., 1995, MolecularMedicine Today 1:174-180]. In addition, when working with mixtures ofcompounds, the risk of losing part of the compounds during theseparation process of the solid phase (filtration or centrifugation) isminimized.

The present invention also contemplates the use of so-callednon-cleavable linkers in connection with such volatilizable solidsupports. A non-cleavable linker is a linker that remains bonded to thecleaved product, but is cleaved from the support. This use leads, aftercleavage, to a modified compound (compound attached to linker) that canbe of interest in itself, or that can be further modified if necessary.

Exemplary non-cleavable linkers can be prepared usingamino-C₂-C₆-alkyl-grafted glass beads as a solid support to prepare acompound such as a peptide. Exemplary aminopropyl glass beads havingdifferent pore sizes, mesh sizes and micromoles of primary amine pergram of glass (μmol/g) are commercially available from Sigma ChemicalCo., St. Louis, Mo., as is aminopropyl silica gel that is said tocontain nitrogen at 1-2 mmoles/g.

Thus, use of aminopropyl-grafted glass beads to form the solid supportlinked peptide, followed by treatment with HF provides a peptide with aC-terminal trifluorosilylpropylamido (—CO—NH—CH₂—CH₂—CH₂—SiF₃) groupthat can be readily hydrolyzed to form the corresponding silicic acidgroup [—CO—NH—CH₂—CH₂—CH₂—Si(OH)₃]. This compound, after partial orcomplete polymerization through the —Si(OH)₃ group, can be used as aconjugate for immunization in the preparation of antibodies against thepeptide of interest. Furthermore, such materials can be useful for theaffinity purification of polyclonal antibodies generated against thepeptide or the compound of interest. The silicon atom can also bepresent after such hydrolyses as a —Si(OH)₂F or —Si(OH)F₂ group, whichcan also be used in a polymerization or other reaction.

In addition to an aminopropyl group, other linking groups are alsocontemplated. For example, 3-mercaptopropyltrimethoxysilane[HS—CH₂—CH₂—CH₂—Si(OCH₃)₃] available from Hüls America, Inc.,Piscataway, N.J. can be coupled to porous glass beads to provide3-mercaptopropyl-grafted glass (thiolated glass). Reaction of thethiolated glass with bis-N-BOC-2-aminoethyl disulfide provides a primaryamine-terminated disulfide after deprotection. The primary amine can beused to synthesize peptides in a usual solid phase synthesis. Uponcompletion of the synthesis, treatment of the reaction product-linkedglass with a reducing agent and then HF provides a peptide having aC-terminal amidoethylmercapto group and a vaporizable remnant of thesolid support. The amidoethylmercapto-terminated peptide can be readilyreacted with an antigenic carrier molecule previously reacted withm-maleimidobenzoyl-N-hydoxysuccinimide ester (ICN Biochemicals, Inc.,Costa Mesa, Calif.) or succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, Pierce ChemicalCo., Rockford, Ill.) to form an immunogenic conjugate.

The disulfide-containing BOC-protected linking group precursor can beprepared by standard techniques. For example, 2-aminoethyl disulfide canbe reacted with two moles of2-(tert-butoxycarbonyl-oxylmino)-2-phenylacetonitrile orN-(tert-butoxy-carbonyloxy)phthalimide or a similar reagent to formbis-N-BOC-2-aminoethyl disulfide.

Several reducing reagents are well known to be useful for breaking thedisulfide bond. Exemplary reagents include sodium borohydride,2-mercapto-ethanol, 2-mercaptoethylamine, dithiothreitol anddithioerythritol. Mercaptan-containing carboxylic acids having two tothree carbon atoms and their alkali metal and ammonium salts are alsouseful. Those reagents include thioglycolic acid, thiolactic acid and3-mercaptopropionic acid. Exemplary salts include sodium thioglycolate,potassium thiolactate, ammonium 3-mercaptopropionate and(2-hydroxyethyl)-ammonium thioglycolate.

The use of cleavable linking groups that separate both from the cleavedproduct and from the solid support is also contemplated. One group ofcleavable linkers contains a benzyl group and silicon. Upon treatmentwith specific reagents, such cleavable linkers can be transformed intogases or liquid forms that can be readily volatilized at various usefultemperatures and pressures. Such linking groups are thus cleavable andform volatile compound(s) on reaction of HF with the solid support.

For example, linkers such as Cl—CH₂C₆H₄—(CH₂)₃₋₅—SiCl₃,Cl—CH₂C₆H₄—(CH₂)₃₋₅—Si(CH₃)Cl₂, Cl—CH₂C₆H₄—(CH₂)₃₋₅—Si(CH₃)₂Cl,Cl—CH₂C₆H₄—SiCl₃ and Cl—CH₂—C₆H₄—Si(OCH₃)₃ can be reacted with glassbeads (or any SiO₂ based material) to form α-chlorobenzylC₃-C₅-alkyl-grafted glass beads or α-chlorobenzyl-grafted glass beads,respectively, that contain one or more siloxane bonds with the support.Exemplary α-cholorbenzyl C₃-C₅-alkyl chlorosilanes and α-chlorobenzylchloro- or methoxysilanes are avaliable from Hüls America, Inc.,Piscataway, N.J. This grafted glass support can then be reacted throughthe chloromethyl group with a wide variety of compounds such asprotected amino acids, amines, alcohols, and the like to form benzylether groups. In the case where n=1 and one methylene group is presentbetween the ring and silicon atom, this linker can be transformed intothe volatile para(trifluorosilylmethyl)benzyl fluoride(F—CH₂C₆H₄—CH₂—SiF₃) by treatment with gaseous or liquid hydrogenfluoride.

Although porous glass or other silica-based solid supports are used asexamples here, it is contemplated that a wide range of polymeric and/orother solid materials can be used in a similar manner. Thus, the desiredsolid-phase synthesized compounds are cleaved from their solid phase,while the spent solid support is completely degraded to volatile byproducts yielding a clear simplification of the overall synthesisprocess.

As one of many examples, the present invention contemplates use of solidphase polymers such as the poly(benzyl ether) shown in Formula A inwhich n is one to greater than 100,000 and X is the reaction productlinked to the support by ester, amide, urethane, urea, amine or otherbond, or a similar polymer containing a cross-linking agent such as1,3,5-trihydroxymethylbenzene.

Upon cleavage with a strong acid or a range of reducing agents such ashydrogen in the presence of palladium acetate or palladium metalhydrogenation catalyst, not only is the bond between the polymer and thedesired compound X cleaved, but also are the bonds that make up thesolid phase polymer itself. Use of hydrogen fluoride as the cleavageagent, provides the volatile compound shown in Formula B as the primaryproduct.

A contemplated poly(benzyl ether) can be prepared by well-knowntechniques. For example, 1,4-benzenedimethanol and a suitable strongbase such as t-butoxide are reacted with a dihalotoluene such asa,a′-dichloro-p-xylene in an appropriate solvent such as ethylene glycoldiethyl or dimethyl ether. The cross-linker is present at zero to about10 weight percent, and more preferably at about 1 to about 5 weightpercent. After polymerization, halomethyl groups can be added to thephenyl rings to provide further places for linkage of the reactionproduct. For example, chloromethyl groups can be added chloromethylatedby reaction of the polymer with chloromethyl methyl ether in thepresence of aluminum chloride or similar Friedel-Crafts catalyst.

As noted previously, it is preferred that the reaction product becleaved from the solid support in a single step. Where hydrogen fluorideis used along with a porous silica support in peptide synthesis, forexample, the addition of HF to a side chain protected support-linkedpeptide can effect deprotection, cleavage of the peptide from thesupport and conversion of the spent silica support into the volatilecompound SiF₄ all in one step, although several different reactions arecarried out in that one step. It is also contemplated that side chaindeprotection be carried out separately, as where trifluoroacteic acid isused for that reaction. It is also contemplated that cleavage of thereaction product from the support be carried out as a separate step asby the use of triethylamine and methanol, followed by reaction with HFto form the cleaved product peptide and SiF₄ that is then removed byvolatilization.

The cleaved product is preferably recovered directly, but is usuallypurified chromatographically prior to further use. However, it is alsocontemplated that the cleaved product can be further reacted withoutrecovery or further purification.

The following Examples are offered to further illustrate, but not limitthe present invention.

EXAMPLE 1 Stability of a Peptide in the Presence of SiliconTetrafluoride

Peptide J21-7 (H-NSSSSQFQIHGPR-OH; SEQ ID NO: 1) was synthesized onMerrifield resin using traditional peptide chemistry (Boc chemistry)with Simultaneous Multiple Peptide Synthesis (Houghten, 1985, Proc.Natl. Acad. Sci. USA, 82:5131-5135). The peptide was then simultaneouslyside-chain deprotected and cleaved from the resin with hydrogen fluoridein the presence and the absence of glass beads to verify theinnocuousness of silicon tetrafluoride towards the peptide. Twodifferent grades of commercially available grafted glass beads were usedfor the experiment (Aminopropyl Glass Beads 80-120 mesh, 77 μmol/g andAminopropyl Glass Beads 200-400 mesh, 152 μmol/g Sigma Chemical Co.).Results are reported in Table 1, below.

TABLE 1 Change in Weight. Solids. After Extraction Weight cleavage (Δ)with AcOH Bag # Content (mg) (mg) (mg) M1 Nothing N/A Total Wt. ofsolids in bag zero −10 ~0 M2 Aminopropyl Glass beads 300 80-120 mesh, 77mmol/g Total Wt. of solids in bag 300 −310 ~0 M5 Aminopropyl Glass beads298 200-400 mesh, 152 mmol/g Total Wt. of solids in bag 298 −292 ~0 M9Peptide resin J21-7 298 HPLC, MS Total Wt. of solids in bag 298 −14562.0 M4 Aminopropyl Glass beads 299 80-120 mesh, 77 mmol/g & Peptideresin J21-7 298 HPLC, MS Total Wt. of solids in bag 597 −448 57.0 M7Aminopropyl Glass beads 297 200-400 mesh, 152 mmol/g & Peptide resinJ21-7 295 HPLC, MS Total Wt. of solids in bag 593 −441 46.0

As is seen from Table 1 above, no weighable residue was recovered whenglass beads alone are treated with HF (Table 1: Bag # M2 and M5). Theweight loss of the bags during cleavage exactly corresponded to theweight of glass beads in the bags plus the weight of the HF labileprotecting groups. No modification of the peptide was observed by massspectroscopy (MS) and high pressure liwuid chromatography (HPLC) whencleaved in the presence of glass beads (Bag M9 compared to bags M4 andM7.)

EXAMPLE 2 Characterization of a Peptide Synthesized on Glass Beads

The peptide H-YGGFLR-NH₂ (SEQ ID NO: 2) was synthesized on two differentgrades of aminopropyl-grafted glass beads [Aminopropyl Glass Beads80-120 mesh, 77 μmol/g (A) and Aminopropyl Glass Beads 200-400 mesh, 152μmol/g (B)] using traditional peptide chemistry (Boc chemistry as inExample 1) in a small reaction vessel fitted with a fritted filter atthe bottom. The peptide was then simultaneously side-chain deprotectedand cleaved from the support with concomitant formation of SiF₄ byliquid hydrogen fluoride. Results are reported in Table 2, below.

TABLE 2 Resin Theor. Actual Molecular Weight Yield Yield Weight(Observ.) Content (mg) (mg) (mg) (Calc) M + H⁺ Δ A H—YGGFLR—NH₂ on 100354.9 36 711 831.9 119.9 Aminopropyl Glass beads 80-120 mesh, 77 μeq/g BH—YGGFLR—NH₂ on 1006 108.7 100 711 831.9 119.8 Aminopropyl Glass beads200-400 mesh, 152 μeq/g —CH₂—CH₂—CH₂—SiF₃: = 127.1 —CH₂—CH₂—CH₂—Si(OH)₃:= 121.1

HPLC traces of the crude material showed the presence of the same mainpeak for both lots. The mass spectral analysis of the main peak observedon the HPLC trace indicates a molecular weight of 831.85. The differenceof 120.85 units compared to the expected molecular weight of 711corroborates the structure H—YGGFLR—NH—CH₂—CH₂—CH₂—Si(OH)₃ for the finalcompound indicating that hydrolysis of the trifluorosilyl group hadoccurred, although a terminal —Si(OH)₂F or —Si(OH)F₂ could also bepresent.

From the foregoing, it will be observed that numerous modifications andvariations can be effected without departing from the true spirit andscope of the present invention. It is to be understood that nolimitation with respect to the specific examples presented is intendedor should be inferred. The disclosure is intended to cover by theappended claims modifications as fall within the scope of the claims.

1. In a solid phase synthesis method wherein a saccharide is coupled toa solid phase support, a plurality of reactions are carried out upon thesolid phase-coupled saccharide to form a solid phase-coupled reactionproduct and that reaction product is cleaved from the solid phasesupport to form a cleaved product, the improvement in which the solidphase support is converted to volatile compound that is separated fromthe cleaved product by vaporization of said volatile compound, leavingbehind said cleaved product.
 2. The solid phase synthesis methodaccording to claim 1 wherein said cleaved product is an oligosaccharide.3. The solid phase synthesis method according to claim 1 whereinreaction product is cleaved from said solid support and the solid phasesupport is converted to a volatile compound in a single step by reactionof the solid phase-coupled reaction product with hydrogen fluoride. 4.The solid phase synthesis method according to claim 1 including thefurther step of recovering the cleaved product.
 5. The solid phasesynthesis method according to claim 1 wherein said solid phase supportis silica.
 6. The solid phase synthesis method according to claim 5wherein said saccharide coupled to said solid phase silica support bymeans of a linking group.
 7. The solid phase synthesis method accordingto claim 6 wherein said silica solid support and linking group isα-chlorobenzyl C₃-C₅-alkyl-grafted glass beads.
 8. The solid phasesynthesis method according to claim 6 wherein said silica solid supportand linking group is amino-C₂-C₆-alkyl-grafted glass beads.
 9. The solidphase synthesis method according to claim 1 wherein said solid phasesupport is a poly(benzyl ether).
 10. The solid phase synthesis methodaccording to claim 9 wherein said solid phase support is shown inFormula A in which n is one to greater than 100,000 and X is thereaction product